Project Abstract Excessive fat accumulation in the liver can lead to liver failure and is a growing health concern. Hepatic steatosis, the first stage of nonalcoholic fatty liver disease (NAFLD), has been reported in one out of every three adults in the United States. Despite the clinical relevance of NAFLD, questions regarding its molecular pathogenesis remain unanswered. A genome wide study for genes linked to NAFLD demonstrated that the I148M variant of PNPLA3 is robustly associated with hepatic triglyceride (TG) content. However, the role of PNPLA3 in the liver and the mechanism for how the I148M variant promotes liver fat accumulation remain elusive. We have found PNPLA3 interacts with ?/? hydrolase domain-containing 5 (ABHD5), the activator of the main TG lipase found in the liver, adipose triglyceride lipase (ATGL; officially PNPLA2). This interaction is dynamic and regulated by nutritional status. Furthermore, the I148M variant increases PNPLA3 affinity for ABHD5. Our central hypothesis is that PNPLA3 interaction with ABHD5 suppresses lipolysis by preventing the interaction of ABHD5 with ATGL/PNPLA2. In addition, we propose that the I148M variant significantly increases affinity with ABHD5, leading to greater suppression of lipolysis and greater liver fat accumulation. This proposal will examine the how PNPLA3 and the I148M variant affect the subcellular trafficking of ABHD5 and ATGL. We will also determine the functional consequences of the interaction of PNPLA3 and the I148M variant with ABHD5 in hepatocytes. The results of this proposal will lead to a better understanding of how ABHD5 and PNPLA3 regulate lipid metabolism in the liver and how the I148M variant disrupts this regulation leading to increased fat accumulation. Completion of this study will provide the trainee with experience and tools needed to pursue a career as a physician-scientist.